Uchida Naoya, Green Rashidah, Ballantine Josiah, Skala Luke P, Hsieh Matthew M, Tisdale John F
Molecular and Clinical Hematology Branch, National Heart Lung and Blood Institute (NHLBI)/National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK), National Institutes of Health (NIH), Bethesda, MD.
Molecular and Clinical Hematology Branch, National Heart Lung and Blood Institute (NHLBI)/National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK), National Institutes of Health (NIH), Bethesda, MD.
Exp Hematol. 2016 Feb;44(2):106-15. doi: 10.1016/j.exphem.2015.10.003. Epub 2015 Oct 21.
Unlike cell lines, human hematopoietic stem cells (HSCs) are less efficiently transduced with HIV-1 vectors, potentially limiting this approach. To investigate which step (internalization, reverse transcription, nuclear transport, and integration) limits lentiviral transduction, we evaluated the kinetics of lentiviral transduction in human CD34(+) cells. We transduced HeLa and CD34(+) cells with self-inactivating HIV-1 vector at low and tenfold higher multiplicity of infection (MOI) and evaluated vector amounts at various time points based on the rationale that if a given step was not limiting, tenfold greater vector amounts would be obtained at the tenfold higher MOI. We observed slower internalization (>60 min), a peak in reverse transcription at 24 hours, and completion of integration at 3 days in CD34(+) cells. In HeLa cells, there were approximately tenfold greater amounts at high MOI at all time points. When compared with HeLa cells, CD34(+) cells exhibited larger differences in vector amounts between high and low MOIs at 2-6 hours and a smaller difference at 12 hours to 10 days, revealing a limitation in human CD34(+) cell transduction around 12 hours, which corresponds to reverse transcription. In serial measurements of reverse transcription at 24 hours, vector amounts did not decrease once detected among CD34(+) cells. When using an HSC expansion medium, we observed less limitation for starting reverse transcription and more efficient transduction among CD34(+) cells in vitro and in xenografted mice. These data suggest that it is the initiation of reverse transcription that limits lentiviral transduction of human CD34(+) cells. Our findings provide an avenue for optimizing human CD34(+) cell transduction.
与细胞系不同，人类造血干细胞（HSC）被HIV-1载体转导的效率较低，这可能会限制这种方法的应用。为了研究哪个步骤（内化、逆转录、核转运和整合）限制了慢病毒转导，我们评估了慢病毒在人类CD34(+)细胞中转导的动力学。我们用自我失活的HIV-1载体以低感染复数（MOI）和高十倍的MOI转导HeLa细胞和CD34(+)细胞，并根据这样的理论在不同时间点评估载体量：如果给定步骤不是限制因素，那么在高十倍的MOI下将获得高十倍的载体量。我们观察到CD34(+)细胞内化较慢（>60分钟），逆转录在24小时达到峰值，整合在3天完成。在HeLa细胞中，在所有时间点高MOI下的载体量大约高十倍。与HeLa细胞相比，CD34(+)细胞在2 - 6小时时高MOI和低MOI之间的载体量差异更大，而在12小时至10天时差异较小，这表明人类CD34(+)细胞转导在大约12小时时存在限制，这与逆转录相对应。在24小时对逆转录的连续测量中，一旦在CD34(+)细胞中检测到载体量就不会减少。当使用造血干细胞扩增培养基时，我们观察到在体外和异种移植小鼠中，CD34(+)细胞起始逆转录的限制较小且转导更有效。这些数据表明，是逆转录的起始限制了人类CD34(+)细胞的慢病毒转导。我们的发现为优化人类CD34(+)细胞转导提供了一条途径。
